The sequential proliferation, lineage-specific differentiation, migration and death of the epithelial cells of the intestinal mucosa is a tightly regulated process modulated by a broad range of regulatory peptides, neurotransmitters, bioactive lipids and differentiation signals. Despite its fundamental importance for understanding intestinal homeostasis, wound healing and pathogenesis of human diseases, the signaling mechanisms involved remain incompletely understood. Many gastrointestinal (GI) peptides, neurotransmitters and bioactive lipids initiate their characteristic effects in their target cells through heptahelical G protein-coupled receptors (GPCRs). Protein kinase D (PKD1) is emerging as a key node in GPCR signaling and consequently the understanding of PKD1 regulation and function is of intense interest and potential impact. The studies proposed here will identify new links between GPCR/PKD1 signaling and pivotal pathways that control migration and proliferation of intestinal epithelial cells. The overarching hypothesis to be explored is that PKD1 plays a critical role in the signal transduction pathways leading to intestinal epithelial cell proliferation. Three Specific Aims are proposed: Specific Aim 1: Characterize the role of PKD1 signaling in intestinal epithelial cell proliferation in vivo and in stem cell-derived intestinal organoids. This Aim will focus on PKD1 signaling in vivo, examining its function in homeostatic turnover via stem/progenitor cell proliferation and repair in response to injury of the intestinal mucosa, using genetically modified mouse models, including PKD1 transgenic mice and stem-cell derived intestinal organoids. Specific Aim 2) Characterize crosstalk mechanisms between PKD1 and -catenin signaling systems in intestinal epithelial cells. The studies proposed in this Aim will characterize an novel crosstalk between GPCR/PKD1 and -catenin signaling, leading to PKD1/- catenin complex formation, -catenin translocation to the nucleus, stimulatory phosphorylation at Ser552 and transcriptional activation; Specific Aim 3) Identify a novel mechanism of PKD1 regulation through PAK-mediated PKD1 phosphorylation at the N-terminal residue Ser203. Based on new preliminary results, we will identify a novel phosphorylation in the N-terminal domain of PKD1 in response to GPCR agonists in intestinal epithelial cells mediated by p21-activated kinases (PAKs), effectors for Rac and Cdc42. The discovery of a PAK/PKD1 cascade uncovers a new point of integration in the signal transduction pathways initiated by GPCR agonists. We anticipate that the mechanistic studies proposed in this application will identify novel upstream pathway(s) and downstream crosstalk mechanisms by which PKD1 regulates the proliferation and migration of intestinal epithelial cells thereby providing the rationale for innovative therapeutic intervention in diseases of the digestive system.